Receptor-mediated endocytosis plays an important role in allowing mammalian cells to sense and respond to their environment. During the last ten years, significant progress has been made in understanding this fundamental process, but a number of questions remain unanswered. The primary goal of the research described in this proposal is to make accurate, temporally-resolved measurements of the related processes of ligand/receptor acidification, exposure to hydrolytic (lysosomal) enzymes, and sorting of ligands and receptors, and to relate these measurements to the biological role of the ligand being studied. The kinetics of acidification and the temperature dependence of both internalization and acidification will be determined for transferrin, epidermal growth factor (EGF), histocompatibility antigen H-2K, and dextran; the kinetics and temperature dependence of entry of endocytosed fluid into compartments containing proteolytic enzymes will be measured using fluorogenic enzyme substrates. Differences in these properties between cell types, or the same cell type under different conditions (such as iron starvation), will also be determined. Flow cytometric analysis and density gradient centrifugation will be used to determine the kinetics with which the pathways of endocytosis of transferrin, EGF, H2K and dextran diverge, and to determine some of the biochemical characteristics of the compartments involved.